A vital challenge that all cells and organisms constantly have to face is numerous harmful DNA lesions in the genome caused by endogenous and environmental agents. It is important that DNA lesions within the highly transcribed genome are dealt with in a specific and efficient manner to prevent stalling of the transcriptional machinery and cell death. A body of evidence indicates RNA polymerase II (Pol II) is a highly selective DNA damage sensor, since it constantly scans the transcribed genome during transcription. Biochemical and genetic studies indicate that the actions of Pol II when encountering DNA damage vary dramatically for different DNA lesions and can be summarized in the following three major categories: Pol II transcription bypass; Pol II backtracking and TFIIS-mediated cleavage; Pol II stalling and initiation of transcription-coupled repair (TCR). However, little is known about the molecular mechanisms by which Pol II senses different DNA damage and signals different downstream processing pathways. We hypothesize that DNA lesions frequently modify bases leading to changes in the interaction patterns between the template DNA, incoming substrate, and Pol II residues. These changes in the interaction patterns, coupled with the chemical nature of DNA lesions, direct Pol II to different processes: bypass, backtracking, and stalling. A clear understanding of the molecular details of the relevant protein-nucleic acid and protein-protein interactions will uncover the mechanisms underlying DNA damage processing pathways. The goal of this research project is to understand how the transcription machinery responds to DNA damage through a multidisciplinary approach that combines structural biology, chemical biology, biophysics, biochemistry, computational biology, and genetics.